GFP and its numerous related fluorescent proteins are now in widespread use as protein tagging agents (for review, see Verkhusha et al., 2003, GFP-like fluorescent proteins and chromoproteins of the class Anthozoa. In: Protein Structures: Kaleidescope of Structural Properties and Functions, Ch. 18, pp. 405-439, Research Signpost, Kerala, India). In addition, GFP has been used as a solubility reporter of terminally fused test proteins (Waldo et al., 1999, Nat. Biotechnol. 17:691-695; U.S. Pat. No. 6,448,087, entitled ‘Method for Determining and Modifying Protein/Peptide Solubility’). GFP-like proteins are an expanding family of homologous, 25-30 kDa polypeptides sharing a conserved 11 beta-strand “barrel” structure. The GFP-like protein family currently comprises some 100 members, cloned from various Anthozoa and Hydrozoa species, and includes red, yellow and green fluorescent proteins and a variety of non-fluorescent chromoproteins (Verkhusha et al., supra). A wide variety of fluorescent protein labeling assays and kits are commercially available, encompassing a broad spectrum of GFP spectral variants and GFP-like fluorescent proteins, including DsRed and other red fluorescent proteins (Clontech, Palo Alto, Calif.; Amersham, Piscataway, N.J.).
GFP fragment reconstitution systems have been described, mainly for detecting protein-protein interactions, but none are capable of unassisted self-assembly into a correctly-folded, soluble and fluorescent re-constituted GFP, and no general split GFP folding reporter system has emerged from these approaches. For example, Ghosh et al, 2000, reported that two GFP fragments, corresponding to amino acids 1-157 and 158-238 of the GFP structure, could be reconstituted to yield a fluorescent product, in vitro or by coexpression in E. coli, when the individual fragments were fused to coiled-coil sequences capable of forming an antiparallel leucine zipper (Ghosh et al., 2000, Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J. Am. Chem. Soc. 122: 5658-5659). Likewise, U.S. Pat. No. 6,780,599 describes the use of helical coils capable of forming anti-parallel leucine zippers to join split fragments of the GFP molecule. The patent specification establishes that reconstitution does not occur in the absence of complementary helical coils attached to the GFP fragments. In particular, the specification notes that control experiments in which GFP fragments without leucine zipper pairs “failed to show any green colonies, thus emphasizing the requirement for the presence of both NZ and CZ leucine zippers to mediate GFP assembly in vivo and in vitro.”
Similarly, Hu et al., 2002, showed that the interacting proteins bZIP and Rel, when fused to two fragments of GFP, can mediate GFP reconstitution by their interaction (Hu et al., 2002, Visualization of interactions among bZIP and ReI family proteins in living cells using bimolecular fluorescence complementation. Mol. Cell 9: 789-798). Nagai et al., 2001, showed that fragments of yellow fluorescent protein (YFP) fused to calmodulin and M13 could mediate the reconstitution of YFP in the presence of calcium (Nagai et al., 2001, Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc. Natl. Acad. Sci. USA 98: 3197-3202). In a variation of this approach, Ozawa at al. fused calmodulin and M13 to two GFP fragments via self-splicing intein polypeptide sequences, thereby mediating the covalent reconstitution of the GFP fragments in the presence of calcium (Ozawa et al., 2001, A fluorescent indicator for detecting protein-protein interactions in vivo based on protein splicing. Anal. Chem. 72: 5151-5157; Ozawa et al., 2002, Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time. Anal. Chem. 73: 5866-5874). One of these investigators subsequently reported application of this splicing-based GFP reconstitution system to cultured mammalian cells (Umezawa, 2003, Chem. Rec. 3: 22-28). More recently, Zhang et al., 2004, showed that the helical coil split GFP system of Ghosh et al., 2000, supra, could be used to reconstitute GFP (as well as YFP and CFP) fluorescence when coexpressed in C. elegans, and demonstrated the utility of this system in confirming coexpression in vivo (Zhang et al., 2004, Combinatorial marking of cells and organelles with reconstituted fluorescent proteins. Cell 119: 137-144).
Although the aforementioned GFP reconstitution systems provide advantages over the use of two spectrally distinct fluorescent protein tags, they are limited by the size of the fragments and correspondingly poor folding characteristics (Ghosh et al., Hu et al., supra), the requirement for a chemical ligation step (Ozawa et al., 2001, 2002 supra), and co-expression or co-refolding to produce detectable folded and fluorescent GFP (Ghosh et al., 2000; Hu et al., 2001, Zhang et al. 2004 supra). Poor folding characteristics limit the use of these fragments to applications wherein the fragments are simultaneously expressed or simultaneously refolded together. Such fragments are not useful for in vitro assays requiring the long-term stability and solubility of the respective fragments prior to complementation. An example of an application for which such split protein fragments are not useful would be the quantitative analysis the interaction of polypeptides tagged with the members of the split protein pair. Another example would be the detection of protein interactions wherein the tagged polypeptides are not simultaneously expressed, or in which interactions are induced after expression by the addition of a small molecule effector such as a drug.
An ideal protein interaction detection system would be genetically encoded, could work both in vivo and in vitro, provide a sensitive analytical signal, and would not require external chemical reagents or substrates. In U.S. Pat. No. 6,428,951 (Michnick et al.), describe various split protein complementation assays for detected protein-protein interactions. However, the split proteins specified are poorly folded and mostly insoluble (see gels of fragments of dihydrofolate reductase). In that application, the fragments of GFP specified are also poorly folded. Michnick et al. describes an approach to improve the folding of the fragments of split proteins wherein the split proteins are fused to known interacting domains, and the split proteins are mutated, and libraries are co-expressed within cells and selected for the function associated with the reconstituted split protein. The DHFR is used as an exemplary case. However, the fact that the specified DHFR fragments used in the claimed embodiment are mostly insoluble when expressed separately, despite being capable of complementation and enzymatic activity when reassembled using fused coiled-coils argues that this directed evolution approach based on co-expression of complementary fragments is not sufficiently stringent to select for soluble and stable fragments. Further, in co-owned, co-pending U.S. patent application Ser. No. 10/973,693 filed Oct. 25, 2004, Waldo et al. demonstrate that co-expression of insoluble split-GFP fragments can lead to complementation, whereas complementation does not occur when the fragments are separately expressed. Waldo et al. further show that a directed evolution using sequential expression of fragments of split proteins can be used to select more soluble, stable versions of split protein fragments. This sequential expression is in marked contrast to the co-expression specified by Michnick et al. A split fluorescent protein tagging system that does not aggregate prior to association and does not change the solubility of the tagged polypeptides has been recently described (Cabantous et. al., 2004, Protein tagging and detection using engineered self-assembling fragments of green fluorescent protein. Nature Biotechnology DOI 10.1038/Nbt1044). However, the fragments are capable of spontaneously self-associating without the need for fused interacting protein domains. Split GFP fragments that remain soluble prior to association, do not change the solubility of fused target proteins, and are also dependent on fused interacting domains for complementation, are needed and are addressed by this invention.